A reflectometry and an ellipsometry are photometic analysis technologies for finding the thickness or optical physical properties of samples by quantifying a change in the reflectivity or a polarization state of reflected light reflected from a surface of the sample and analyzing the quantified value. Quantifying equipment using the above technologies includes a reflectometer and an ellipsometer. The reflectometer and the ellipsometer are being utilized to evaluate the thin film thickness and the physical properties of various nano levels in the nano thin film manufacturing process of the semiconductor industry. Furthermore, the use of the reflectometer and the ellipsometer is expanded to the bio industry, and an attempt to analyze the interface of bio materials, such as protein, DNA, virus, and new medicine materials, is being made.
A conventional reflectometer is problematic in that it is sufficient to evaluate the thickness and the physical properties of a nano thin film of several nano meters (nm) or higher in size, but it has low reliability because of low measurement sensitivity in analyzing small molecular bio materials requiring the sensitivity of approximately 1 to 0.001 nm range. The ellipsometer has the measurement sensitivity of 0.01 nm or lower, as compared with the reflectometer. In particular, the ellipsometer has high measurement sensitivity in a condition in which the contrast of a refractive index is high as in the thickness measurement of an oxide film having a relatively low refractive index than a semiconductor formed over a semiconductor substrate of a high refractive index.
However, the ellipsometer requires a quantifying method with an improved sensitivity in order to analyze even small molecular bio materials.
A conventional technique for improving the measurement sensitivity when analyzing bio materials includes a Surface Plasmon Resonance (SPR) sensor (hereinafter referred to as an ‘SPR sensor’) in which the reflectometry and a surface plasmon resonance technology are mixed. A surface plasmon resonance phenomenon refers to a phenomenon in which electrons existing in a metal surface are excited by light waves and collectively vibrated in the normal direction of the surface and, at this time, light energy is absorbed. The SPR sensor has been known to be able to measure the thickness and a change in the reflective index of a nano thin film adjacent to a metal surface by using the surface plasmon resonance phenomenon sensitive to the polarization characteristic of light and also to measure a change in the binding concentration of a bio material by using a non-labeling method not using a fluorescent material in real time.
FIG. 1 is a diagram showing the construction of a conventional SPR sensor for analyzing bio materials. As shown in FIG. 1, the conventional SPR sensor chiefly includes a prism 10, a thin metal film 20, a micro flow path 30, a light source 40, a polarizer 50, an analyzer 60, and a photodetector 70. The conventional SPR sensor has the thin metal film 20, such as gold (Au) or silver (Ag), coated on one surface of the prism 10 in thickness of several tens of nm and has the micro flow path 30 formed on the thin metal film 20. Here, when a buffer solution 34 in which samples 32 of bio materials are dissolved is injected into the micro flow path 30, the bio materials are bound to ligand materials 22 formed on a surface of the thin metal film 20, thus forming a binding layer of a predetermined thickness.
Next, light generated by the light source 40 is polarized by the polarizer 50. The polarized incident light is incident on the interface of the thin metal film 20 at a Surface Plasmon Resonance angle (hereinafter referred to as an SPR angle (spr)) via the prism 10 so that it generates surface plasmon resonance is generated. Here, the reflected light reflected from the thin metal film 20 includes optical data regarding the binding layer of the samples 32. That is, in the process of the samples 32 being bound to and dissociated from the thin metal film 20, the binding and dissociation kinetics of molecular interactions, such as a binding concentration and the thickness or refractive index of the binding layer, are changed, and thus a surface plasmon resonance condition is changed.
FIG. 2 shows a binding curve appearing in the process of the samples 32 being bound to the thin metal film 20 and a dissociation curve appearing in the process of the samples 32 being dissociated from the thin metal film 20. In FIG. 2, a rise in the association rate constant ka means that fast absorption of the bio materials, and a fall in the dissociation rate constant kd means that the bio materials are slowly dissociated. In other words, a dissociation constant KD=kd/k3 of an equilibrium state can be found by measuring the association rate constant and the dissociation rate constant. For example, it can be determined whether a small molecular and new medicine candidate material that can be used as a carcinogenic agent can be used as a new medicine by measuring a characteristic that the small molecular and new medicine candidate material is bound to or dissociated from protein including cancer risk factors.
Next, the reflected light including optical data, such as that described above, is detected by the photodetector 70 via the prism 10 and the analyzer 60. Here, the photodetector 70 can find the binding and dissociation kinetics of molecular interactions of the samples 32 by measuring a change in the polarized components of the reflected light (that is, the intensity of the reflected light).
Problems of the conventional SPR sensor for analyzing bio materials are described below with reference to FIGS. 3 and 4. FIG. 3 is a graph showing the measurement of the ellipsometric constant Ψ using the SPR sensor, which shows a similar characteristic to the conventional reflectivity. As shown in FIG. 3, the thin metal film 20 was formed of a thin Au film of 50 nm in thickness, and the light source 40 having a wavelength of 633 nm was used. Furthermore, a binding layer was measured 0 nm and 1 nm in thickness. Furthermore, the binding layer was measured 1.45 in the refractive index n, and the buffer solution 34 was measured 1.333 and 1.334 in the refractive index n.
In the principle of the conventional SPR sensor, the amount of a shift in the SPR angle according to time, showing a minimum reflectivity, is measured by measuring the reflectivity or the ellipsometric constant Ψ from which a change in the intensity of reflected light can be known. Here, if the surface plasmon resonance phenomenon is satisfied, the reflectivity or the ellipsometric constant Ψ has a minimum value, and the SPR angle is near 59° as shown in FIG. 3. It can also be seen that the ellipsometric constant Ψ moves to the right with an increase of the thickness of the binding layer and also a rise in the refractive index of the buffer solution 34. FIG. 3 shows a comparison of a case in which bio materials having a refractive index of 1.45 bound about 1 nm and a case in which there was no binding of bio materials and there was a change in the SPR angle when only the refractive index of the buffer solution 34 was changed from 1.333 to 1.334. From FIG. 3, it can be seen that the two cases show a similar change in the SPR angle. In other words, only pure binding and dissociation characteristics from which a change in the reflective index of the buffer solution has been removed must be measured, but it can be seen that when the binding and dissociation characteristics of bio materials are measured, a problem arises in the measurement results because of a change in the reflective index of the buffer solution.
FIG. 4 is a diagram illustrating a conventional problem in which the binding and dissociation kinetics inherent in samples, appearing in a process of the samples being bound and dissociated, and a change in the refractive index resulting from a buffer solution are mixed together. FIG. 4 is a graph showing the binding and dissociation concentrations inherent in the samples 32 appearing in the binding and dissociation process. FIG. 5 is a graph showing a change in the measurement results of the SPR sensor resulting from a change in the reflective index of the buffer solution 34. FIG. 6 is a graph showing the binding and dissociation concentrations of the samples 32, measured by the SPR sensor, in the state in which the binding concentration inherent in the samples 32 and a change in the refractive index resulting from the buffer solution 34 are mixed together. That is, the samples 32 are very sensitive to effects (indicated by arrows) according to a change in the reflective index of the buffer solution 34, and thus the binding and dissociation concentrations of only pure samples 32 do not clearly appear. Accordingly, it is difficult to calculate the binding and dissociation concentrations of the samples 32 by analyzing the binding and dissociation concentrations of only pure samples 32.
On the other hand, in order to correct a change in the reflective index of the buffer solution 34 and to prevent errors resulting from diffusion between the samples 32 and the buffer solution 34, a correction method using an elaborate valve apparatus and an elaborate air injection apparatus and two or more channels used as a reference channel is being used. However, this method is difficult to distinguish a change in the SPR angle resulting from a change in the reflective index of the buffer solution 34 and a change in the SPR angle resulting from pure binding and dissociation characteristics, and the changes can always serve as measurement error factors. Consequently, the conventional SPR sensor has a fundamental problem in measuring the binding and dissociation characteristics of a material having a low molecular weight, such as small molecule, because of the limits of a measurement method, such as that described above.
Furthermore, the conventional SPR sensor requires a high manufacturing cost for the sensor because it uses the thin metal film 20 made of a noble metal, such as gold (Au) or silver (Ag), for surface plasmon resonance. In addition, the thin metal film 20 is problematic in that a refractive index has a severe variation because surface roughness is not uniform according to a manufacturing process and quantitative measurement for bio materials are difficult because of an unstable optical characteristic.